The present invention relates to methods for determining concentrations of lipoproteins and lipids in blood and, more particularly, to such methods which utilize turbidimetric measurement.
Lipoproteins are aggregates of lipids and protein which circulate in the blood and are the means by which lipids are transported within the body. The lipid portions of these aggregates consist essentially of cholesterol and triglyceride. The various classes of lipoproteins contain cholesterol and triglyceride in different proportions and from different sources. Thus, by determining the concentrations of the various lipoprotein classes in blood, one can determine the concentrations of cholesterol and triglyceride. Since the concentrations of both lipoproteins and lipids correlate with the incidence of various circulatory and coronary diseases, they are of great medical importance.
Lipoproteins are divided into four major classes based on their behavior in prior art methods of analysis. These classes are: chylomicrons (abbreviated herein as CHYLO), very low density lipoproteins (also known as pre-beta lipoproteins and abbreviated herein as VLDL), low density lipoproteins (also known as beta lipoproteins and abbreviated herein as LDL) and high density lipoproteins (also known as alpha lipoproteins and abbreviated herein as HDL). Minor classes such as, for example, floating beta lipoproteins, lipoprotein X, sinking pre-beta lipoproteins, and the like occur less frequently. The presence of an abnormally high level of certain of these classes of lipoproteins has been correlated with various disorders, conveniently expressed according to the system of Frederickson, et al. In this classification system, the presence of various clinical disorders is related to elevated levels of various classes of lipoproteins. By determining the concentrations of the lipoprotein classes in blood, one can therefore detect the presence of these disorders. The Frederickson classification system is summarized in Table I.
TABLE I ______________________________________ The Major Abnormal Lipoprotein Patterns.sup.(a) Type Chylomicrons LDL VLDL Floating beta Lipoproteins ______________________________________ I + IIa + IIb + + III + IV + V + + ______________________________________ .sup.(a) "+" indicates elevated concentration.
In the past, measurement of total cholesterol and total triglyceride has been used to detect certain of these disorders. But because the various classes of lipoproteins contain cholesterol and triglyceride in different proportions as discussed above, determination of only total cholesterol and total triglyceride is not sufficient to differentiate among the six major abnormal lipoprotein patterns discussed above. It is, therefore, necessary to determine the concentrations of lipoproteins rather than just lipids in order unequivocally to determine the types of abnormal lipoprotein patterns described by Frederickson, et al., and thus the associated disorders.
Furthermore, determination of lipoprotein concentrations allows more convenient calculation of lipid concentrations than with prior art methods. Prior art methods for determination of cholesterol and triglyceride concentrations require the subject to fast from ten to fourteen hours prior to withdrawal of the sample. This is a disadvantage, as discussed below. Further, prior art methods generally require extraction of the serum with various solvents to enhance their specificity. This requirement imposes an additional step on prior art lipid determination methods and is seen to be a further disadvantage. Moreover, no rapid screening technique for accurately determining the sum of cholesterol and triglyceride concentrations is known.
Three techniques have generally been used in the prior art to determine the concentrations of the various lipoprotein classes: visual evaluation of the plasma, electrophoresis, and ultracentrifugation. All three techniques generally require the subject to fast for ten to fourteen hours prior to the drawing of the sample to minimize the effect of exogenous chylomicrons on lipid measurements. It is a disadvantage of these techniques that they require this fasting, which is a hardship to the patient and makes him less willing to undergo these tests.
Visual evaluation of the plasma is not a quantative technique but is of use in detecting chylomicrons. When plasma stands in a tube at 0-4.degree. C. for 18-24 hours, the chylomicrons will rise to the top of the tube and are visible as a layer of "cream." Because of its lack of precision, this technique is unsatisfactory unless combined with another.
Electrophoresis is a relatively inexpensive and simple method compared to ultracentrifugation but has several serious disadvantages. It is at best semiquantitative, and unequivocal interpretation often requires total serum cholesterol and triglyceride analysis. Therefore, the method is not independent and must be supplemented by other measurements. Also, quantification of the lipoprotein electrophoresis bands is a difficult procedure, whether done by chemical determination or by densitometry.
While ultracentrifugation is a more easily quantified method for determination of the individual lipoprotein classes, it is both expensive and time-consuming. Because of the expense, it is not generally possible for blood analysis to be performed using this method by the physician himself or by one in his employ. In fact, this technique is generally not used clinically but only as a research tool, because of its difficulty and because of the expensive equipment required.
The difficulties attendant on the methods of electrophoresis and ultracentrifugation have led to the development of simpler turbidimetric methods.
For example, Stone, et al., Clin. Chim. Acta, 31 (1971) 333-354, have proposed measuring the turbidities of chylomicrons and VLDL in serum by nephelometry to obtain their concentrations. From these lipoprotein concentrations and a measurement of total serum cholesterol, the authors estimate the concentration of LDL. This method has the advantage of being rapid and simple, and does not require fasting by the subject. However, there are many sources of error, including minute scratches on the surface of the measuring cuvette and non-uniform standards.
Scholnick, et al., have also proposed a turbidimetric method for detection and identification of types of hyperlipoproteinema (Protides of the Biological Fluids, Proceedings of the 19th Coloquiuum 1971, H. Peeters, Ed., pp. 289-292). Scholnick, et al., teach a method based on the selective precipitation of lipoproteins by heparin in the presence of divalent cations. In this prior art method, three samples of serum from the blood of the patient under study are prepared and each is separately combined with a certain quantity of heparin (a sulfated polysaccharide), a divalent cation (Mg++ or Ca++), and (for certain samples) sodium ion. The procedure is described by Scholnick, et al., as follows:
"Two ml. of salt solution is added to each of three tubes. To tube one, 0.025 M CaCl.sub. 2, to tube two, 0.1 M MgCl.sub.2 in 0.7% NaCl, and to tube three, 0.1 M MgCl.sub.2 in 1% NaCl. To each of these is added 0.2 ml. of serum and the blank is read at 650 nm in a Coleman Jr. spectrophotometer. To tube one is added 0.01 ml. of 1% heparin in normal saline and to tubes two and three, 0.04 ml. of 5% heparin in normal saline. After the addition of heparin, the tubes are mixed and after 4 min. at room temperature, the turbidity is measured."
The turbidity in sample one is due to the insoluble complexes of chylomicrons, VLDL, and LDL; that in the second sample is due to the insoluble complexes of chylomicrons and VLDL; and that in the third sample is due to the insoluble complex of chylomicrons along. Scholnick, et al., have correlated these turbidities with the abnormal lipoprotein patterns of Frederickson, et al., in an attempt to provide a method for detecting types of hyperlipoproteinemia.
While the method of Scholnick, et al., is to be preferred over the three older techniques previously discussed, it also has certain disadvantages. First, sulfated polysaccharides in general and heparin in particular are expensive and scarce materials. The fact that more than 0.08 ml. of 5% heparin is needed to analyze one blood sample by this prior art method is seen to be a disadvantage. Second, three separate samples of serum are required for each analysis; this increases the manipulations required to prepare for the analysis and also increases the amount of blood which must be obtained to perform the analysis. Third, Scholnick, et al., teach no method for relating the turbidity measurements obtained by their technique to the concentrations of the various individual lipoprotein classes or to the sum of cholesterol and triglyceride concentration in the blood sample. That is, although a linear regression based on data from their laboratory shows a correlation between turbidity measurements obtained through their technique and independently-measured cholesterol or triglyceride concentrations, no method for accurately determining the concentration of individual lipoprotein classes is taught. There is also no teaching of a method for accurately determining the sum of cholesterol and triglyceride concentrations by a single measurement. This latter determination is of great value in screening patients for elevated levels of either material. Further, Scholnick, et al., teach no standardization technique so that a single set of turbidities measured on a given spectrophotometer can be used to determine lipoprotein concentrations.
It is, therefore, an object of the present invention to provide a turbidimetric method for determining concentrations of individual liproprotein classes in blood. It is a further object to provide such a method which uses a minimum of sulfated polysaccharide. It is a further object to provide such a method which uses a minimum of blood serum. It is a further object to provide such a method which involves only a single serum sample and thus a minimum of manipulation and equipment. It is a further object to provide such a method which does not require fasting by the subject prior to withdrawal of the blood sample. It is a further object to provide such a method whereby the sum of the cholesterol and triglyceride concentrations may be determined by a single measurement, thus permitting rapid screening for individuals with excessive levels of either material. It is also an object of the present invention to provide a turbidimetric method for determination of individual lipid concentrations in blood. It is still a further object to provide a standardization technique for relating the turbidimetric measurements obtained on a given spectrophotometer to the concentrations of the various lipoprotein classes or lipids.